scholarly journals Identification of Protein-Protein Interactions and Topologies in Living Cells with Chemical Cross-linking and Mass Spectrometry

2008 ◽  
Vol 8 (3) ◽  
pp. 409-420 ◽  
Author(s):  
Haizhen Zhang ◽  
Xiaoting Tang ◽  
Gerhard R. Munske ◽  
Nikola Tolic ◽  
Gordon A. Anderson ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Protein Interactions ◽  
Living Cells ◽  
Cross Linking ◽  
Protein Protein Interactions ◽  
Chemical Cross Linking

scholarly journals Exploring the Interactome of Cytochrome P450 2E1 in Human Liver Microsomes with Chemical Cross-Linking Mass Spectrometry

2021 ◽  
Author(s):  
Dmitri R. Davydov ◽  
Bikash Dangi ◽  
Guihua Yue ◽  
Bhagwat Prasad ◽  
Viktor G. Zgoda
Keyword(s):  
Mass Spectrometry ◽  
Cytochrome P450 ◽  
Protein Interactions ◽  
Human Liver ◽  
Liver Microsomes ◽  
Human Liver Microsomes ◽  
Cross Linking ◽  
Cytochrome P450 2E1 ◽  
Protein Protein Interactions ◽  
Chemical Cross Linking

This study aimed on exploration of the system-wide effects of the alcohol-induced increase in the content of cytochrome P450 2E1 (CYP2E1) in the human liver on drug metabolism. Using membrane incorporation of purified CYP2E1 modified with photoreactive crosslinkers benzophenone-4-maleimide (BPM) and 4-(N-succinimidylcarboxy)benzophenone (BPS), we explored the array of its protein-protein interactions (proteome) in human liver microsomes (HLM) with chemical cross-linking mass spectrometry (CXMS). Exposure of bait-incorporated HLM samples to light was followed by isolation of the His-tagged bait protein and its cross-linked aggregates on Ni-NTA agarose. Analyzing the individual bands of SDS-PAGE slabs of thereby isolated protein with the toolset of untargeted proteomics, we detected the cross-linked dimeric and trimeric complexes of CYP2E1 with other drug-metabolizing enzymes. Among the most extensively cross-linked partners of CYP2E1 are cytochromes P450 2A6, 3A4, 2C9, and 4A11. We also detected the conjugates of CYP2E1 with UDP-glucuronosyltransferases (UGTs) 1A6, 1A9, 2B4, 2B15, and 2B17. These results demonstrate the exploratory power of the proposed CXMS strategy and corroborate the concept of tight functional integration in the human drug-metabolizing ensemble through protein-protein interactions of the constituting enzymes. Of particular interest is the observation of efficient cross-linking of CYP2E1 with CYP4A11. This enzyme plays a central role in the synthesis of vasoactive eicosanoids and its interactions with alcohol-inducible CYP2E1 may shed light on the mechanisms of alcohol-induced hypertension.

scholarly journals Quaternary Structure of the Tryptophan Synthase α-Subunit Homolog BX1 from Zea mays

2019 ◽  
Author(s):  
Andrew Norris ◽  
Florian Busch ◽  
Michael Schupfner ◽  
Reinhard Sterner ◽  
Vicki Wysocki
Keyword(s):  
Mass Spectrometry ◽  
Protein Interactions ◽  
Quaternary Structure ◽  
Early Time ◽  
Cross Linking ◽  
Tryptophan Synthase ◽  
Protein Protein Interactions ◽  
Α Subunit ◽  
Primary And Secondary Metabolism ◽  
Chemical Cross Linking

The manuscript describes the use of chemical cross-linking/mass spectrometry and mutagenesis to investigate the dimeric interface of the tryptophan synthase α-subunit homolog, BX1. This work indicates that BX1 homodimerization might have served as a mechanism to exclude an interaction with the tryptophan synthase β-subunit, TrpB, at an early time in evolution, thereby eliminating cross-talk between primary and secondary metabolism. This work would be of interest to mass spectrometrists and structural biologist as it presents a workflow to determine the physiological protein-protein interactions within crystal structures using chemical cross-linking/mass spectrometry and mutagenesis as complementary structural biology techniques, thereby eliminating ambiguity and potential mis-assignments due to the presence of additional (artificial) protein contacts formed during the crystallization process.

scholarly journals Quaternary Structure of the Tryptophan Synthase α-Subunit Homolog BX1 from Zea mays

2019 ◽  
Author(s):  
Andrew Norris ◽  
Florian Busch ◽  
Michael Schupfner ◽  
Reinhard Sterner ◽  
Vicki Wysocki
Keyword(s):  
Mass Spectrometry ◽  
Protein Interactions ◽  
Quaternary Structure ◽  
Early Time ◽  
Cross Linking ◽  
Tryptophan Synthase ◽  
Protein Protein Interactions ◽  
Α Subunit ◽  
Primary And Secondary Metabolism ◽  
Chemical Cross Linking

The manuscript describes the use of chemical cross-linking/mass spectrometry and mutagenesis to investigate the dimeric interface of the tryptophan synthase α-subunit homolog, BX1. This work indicates that BX1 homodimerization might have served as a mechanism to exclude an interaction with the tryptophan synthase β-subunit, TrpB, at an early time in evolution, thereby eliminating cross-talk between primary and secondary metabolism. This work would be of interest to mass spectrometrists and structural biologist as it presents a workflow to determine the physiological protein-protein interactions within crystal structures using chemical cross-linking/mass spectrometry and mutagenesis as complementary structural biology techniques, thereby eliminating ambiguity and potential mis-assignments due to the presence of additional (artificial) protein contacts formed during the crystallization process.

Sign in / Sign up

Export Citation Format

Share Document